Suppression of Protein Kinase C?? Triggers Apoptosis Through Down-Regulation of BCL-XL in a Rat Hepatic Epithelial Cell Line

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Abstract
Inactivation of protein kinase C (PKC)α plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKCα inactivation mediates the apoptosis, PKCα was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKCα and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKCα and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKCα were decreased by 54.5% and 41.4%, respectively, after PKCα antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKCα-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKCα and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKCα declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKCα plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.